A layer-by-layer deposited ultrathin film could be fabricated with two kinds of poly(ethyleneimine) based polymers, PEI-Ser-Ade and PEI-Ser-Thy, which have adenine groups and thymine groups, respectively, in the side chains of the polymers. The result of UV-vis absorption measurement showed that the multilayered structure could be fabricated by dipping the substrate alternatively into the solutions. The pH value of the solutions used for the layer-by-layer deposition was one of the important factors determining whether the polymers could be successively adsorbed or not. The in situ surface plasmon measurements revealed that both PEI-Ser-Ade and PEI-Ser-Thy were adsorbed on the PEI-Ser-Ade layer, indicating that the hydrophobic interaction between these polymers contributed to the formation of the layer-by-layer deposited film.